Shuichiro Kobayashi

Injectable and photocross-linkable gels based on gellan gum methacrylate: A new tool for biomedical application

In this work, a natural polysaccharide gellan gum (GG) has been modified with methacrylic groups (GG-MA) and combined with polyethylene glycol dimethacrylate (PEG-DMA) in order to create novel injectable hydrogels that can be easily delivered through a needle and photocross-linked in the injection site. A novel synthetic procedure for methacrylation of GG has been proposed to better control its derivatization. Different degrees of functionalization have been achieved and their effects on the solubility and mechanical properties of GG-MA were investigated. A good balance in terms of hydrophilicity and elasticity of the corresponding hydrogels was identified, although not suitable enough as injectable material for the treatment of damaged soft tissues. For this reason, several concentrations and different molecular weights of PEG-DMA were investigated to modulate the composition of GG-MA hydrogels and overcome their extreme fragility. Swelling abilities of the hydrogels in different media were studied as a key parameter able to affect the release profile of loaded therapeutic agents. Model molecules having different spherical hindrance (sulindac and vitamin B12) were then chosen to study how the hydrogels were able to modulate their diffusion profiles over time. Finally, the hydrogels safety was evaluated trough an MTT cytotoxicity test on human fibroblasts.

Inflammatory Bone Loss in Experimental Periodontitis Induced by Aggregatibacter actinomycetemcomitans in Interleukin-1 Receptor Antagonist Knockout Mice

ABSTRACT The interleukin-1 receptor antagonist (IL-1Ra) binds to IL-1 receptors and inhibits IL-1 activity. However, it is not clear whether IL-1Ra plays a protective role in periodontal disease. This study was undertaken to compare experimental periodontitis induced by Aggregatibacter actinomycetemcomitans in IL-1Ra knockout (KO) mice and wild-type (WT) mice. Computed tomography (CT) analysis and hematoxylin-and-eosin (H&E) and tartrate-resistant acid phosphatase (TRAP) staining were performed. In addition, osteoblasts were isolated; the mRNA expression of relevant genes was assessed by real-time quantitative PCR (qPCR); and calcification was detected by Alizarin Red staining. Infected IL-1Ra KO mice exhibited elevated (P, <0.05) levels of antibody against A. actinomycetemcomitans, bone loss in furcation areas, and alveolar fenestrations. Moreover, protein for tumor necrosis factor alpha (TNF-α) and IL-6, mRNA for macrophage colony-stimulating factor (M-CSF), and receptor activator of NF-κB ligand (RANKL) in IL-1Ra KO mouse osteoblasts stimulated with A. actinomycetemcomitans were increased (P, <0.05) compared to in WT mice. Alkaline phosphatase (ALP), bone sialoprotein (BSP), osteocalcin (OCN)/bone gla protein (BGP), and runt-related gene 2 (Runx2) mRNA levels were decreased (P, <0.05). IL-1α mRNA expression was increased, and calcification was not observed, in IL-1 Ra KO mouse osteoblasts. In brief, IL-1Ra deficiency promoted the expression of inflammatory cytokines beyond IL-1 and altered the expression of genes involved in bone resorption in A. actinomycetemcomitans-infected osteoblasts. Alterations consistent with rapid bone loss in infected IL-Ra KO mice were also observed for genes expressed in bone formation and calcification. In short, these data suggest that IL-1Ra may serve as a potential therapeutic drug for periodontal disease.

Serum Amyloid A Promotes E-Selectin Expression via Toll-Like Receptor 2 in Human Aortic Endothelial Cells

Periodontitis is a chronic inflammatory disease that affects the periodontium. Recent studies suggest an association between periodontal and cardiovascular diseases. However, the detailed molecular mechanism is unknown. A previous study has demonstrated that experimental periodontitis induces serum amyloid A (SAA) in the liver and peripheral blood of ApoE-deficient mice as an atherosclerosis model. SAA is an acute-phase protein that affects systemic inflammation. The aim of this study is to investigate the atherosclerosis-onset mechanism using human aortic endothelial cells (HAECs) stimulated by SAA in vitro. Atherosclerosis PCR array and qPCR analyses showed upregulation of adhesion molecules such as intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin in HAECs upon SAA stimulation. In addition, the results demonstrated that Toll-like receptor, TLR2, could serve as an important receptor of SAA in HAECs. Furthermore, small interfering RNA (siRNA) against TLR2 inhibited the upregulation of adhesion molecules in HAECs stimulated by SAA. Our results suggest that SAA stimulates the expression of adhesion molecules via TLR2. SAA could be an important molecule for atherosclerosis induced by periodontal disease.

Adhesion Properties of Human Oral Epithelial-Derived Cells to Zirconia.

BACKGROUND Few studies have examined epithelial attachment to zirconia and the proliferative ability of epithelial cells on zirconia surfaces. PURPOSE To evaluate the adhesion properties of zirconia materials for epithelial cell attachment and compare this with titanium and alumina. MATERIALS AND METHODS Human oral epithelial cells were cultured on smooth-surfaced specimens of commercially pure titanium (cpTi), ceria-stabilized zirconia/alumina nano-composite (P-NANOZR), yttria-stabilized zirconia (Cercon), and alumina oxide (inCoris AL). The cell morphology, the cell viability and mRNA of integrin β4 , laminin γ2 , catenin δ2 , and E-cadherin were evaluated by SEM, Cell-Counting Kit-8, and real-time PCR, respectively. RESULTS Morphology of cells attached to specimens was similar among all groups. The viable cell numbers on Cercon and inCoris AL after 24 hours culture were significantly higher than for cpTi. Integrin β4 , laminin γ2 , and catenin δ2 mRNA expression was not different among all groups. However, at 3 and 24 hours after incubation, E-cadherin mRNA expression in the P-NANOZR group was significantly higher than for cpTi. CONCLUSION Zirconia may support binding of epithelial cells through hemidesmosomes comparable with titanium. Furthermore, P-NANOZR may impart resistance to exogenous stimuli through strong intercellular contacts with peri-implant mucosal cells when used as an abutment and implant superstructure.

Canola and hydrogenated soybean oils accelerate ectopic bone formation induced by implantation of bone morphogenetic protein in mice

Highlights • BMP-induced bone formation was evaluated in mice fed a Soy, Can, or H2-Soy diet.• Ectopic bone formation was greater in the Can and H2-Soy groups.• Altered levels of VK2 and VK-dependent protein(s) might affect the bone formation.

Age-related changes of CD4+ T cell migration and cytokine expression in germ-free and SPF mice periodontium

OBJECTIVE Increasing age is a potential risk factor for periodontal tissue breakdown, which may be affected by commensal flora. The aim of this study evaluated age-related changes in CD4+ T cells, C-C chemokine ligand 5 (CCL5), interleukin (IL)-17A, and receptor activator of nuclear factor-kappa B ligand (RANKL) expression using germ-free (GF) and conventionally reared (SPF) mice. DESIGN GF and SPF mice at 8 (n = 6/group) and 22 weeks old (n = 6/group) were used. Immunohistochemical analyses were performed to determine the effects of aging on protein expression in periodontal tissues. Age-related changes in alveolar bone were quantified using micro-CT analysis. RESULTS SPF mice, but not GF mice, showed an age-related increase in alveolar bone loss (P < 0.01). SPF mice at 22 weeks of age increased expression of CD4+ T cells, CCL5, IL-17A, and RANKL compared to those at 8 weeks of age in connective tissue and alveolar bone surface (P < 0.01). Furthermore, there was increased CD4+ T cells, which were co-expressed with IL-17A and RANKL in SPF mice at 22 weeks of age. On the other hand, the GF mice did not show any significant differences in CD4+ T cells, CCL5, IL-17A and RANKL expression between the two age groups. CONCLUSIONS SPF mice induced an age-related increase in CD4+ T cells co- expressed with IL-17A and RANKL, with occurring alveolar bone loss. In contrast, GF mice did not show age-related changes in CD4+ T cell migration and cytokine expression.

Rheological approach for determining yield stresses in flowable resin composites prior to setting

The purpose of this study is to develop a method for quantifying the fluidity of flowable resin composites using determinations of yield stress. Five commercially available composites (AliteFlo LV, Flow-it ALC, Venus flow, Tetric N-flow, Revolution Formula2) were investigated. Yield stress values were obtained by plotting shear stresses for a range of shear rates, followed by fitting of Casson fluid models to flow curve data and extrapolation to the stress axis. To confirm that yield stress reflected fluidity, apparent viscosity at the lower shear rate (0.2 s-1) was calculated from flow curves. Yield stresses ranged from 5.4 to 43.1 Pa, and were found to capture differences in the fluidity of composites that were not captured by viscosity measurement at the low shear rate. Yield stress is directly proportional to fluidity, and could serve as a simple and precise indicator for selecting flowable resin composites for use in various clinical applications.